The Bone Marrow Environment Promotes Resistance to Mitoxantrone and Etoposide By Distinct Mechanisms in Pediatric AML

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. One possible mechanism of chemoresistance is enhanced DNA damage repair. Mitoxantrone and etoposide are standard chemotherapy for AML, both leading to DNA damage by inhibition of topoisomerase II. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main processes for DNA damage repair, with ataxia-telangiectaxia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK) as key components, respectively. Both kinases phosphorylate histone H2AX (gH2AX), which facilitates DNA damage repair. Additionally, the bone marrow stromal environment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. To study leukemia-stroma interactions, we used HS5 and HS27A human bone marrow stromal cells. In co-culture studies, we found that stroma-mediated resistance to mitoxantrone was mediated by both stromal soluble factors and cell-cell contact, whereas resistance to etoposide mainly by physical contact with stroma. Further, we recently reported that stromal CYR61 promotes resistance to mitoxantrone, but not etoposide (Long, et al, 2015, Br J Haematol, 170:704).

To further study the mechanism underlying stroma-induced chemotherapy resistance, 44 diagnostic AML patient samples from the Children's Oncology Group were co-cultured on stromal cells, or cultured alone. The samples were treated with 100 nM mitoxantrone (n=27) or 10 µM etoposide (n=32) for 24h. Fifteen samples had sufficient cells for both chemotherapy treatments. Cells were analyzed by FACS, and stromal cells, which express mOrange, and lymphocytes (CD45^high, SSC^low) were excluded. We measured intracellular levels of cleaved PARP (cPARP) as an apoptosis marker, and gH2AX as a DNA damage signaling marker. As expected, AML cell viability (%cPARP-) after etoposide treatment was significantly higher in the stromal co-cultures (61.2 ±3.3% for AML cells cultured alone, v. 83.2 ±1.8% in HS5 co-cultures, p<0.0001). Results were similar for mitoxantrone treatment. We also found increased DNA damage signaling (%cPARP-/gH2AX+) after mitoxantrone treatment for AML cells in stromal co-culture (34.5 ±3.0% in cells cultured alone, v. 58.6 ±3.1% in HS5 co-cultures, p<0.0001). However, DNA damage signaling was not significantly increased after etoposide treatment. For 5 samples treated with both chemotherapy agents, we also measured pDNA-PKcs and pATM by FACS. Treatment with mitoxantrone, but not etoposide, induced more pDNA-PKcs (MFI, 25.7 ±4.2 untreated, v. 61.7 ±5.1 mitoxantrone, p<0.001, v. 31.5 ±4.8 etoposide) in AML cells cultured alone. However, stroma did not further increase pDNA-PKcs. These results suggest that the NHEJ pathway is important for the repair of DNA damage caused by mitoxantrone, and that stromal cells increase DNA damage signaling by a mechanism not involving increased pDNA-PKcs.

To better understand etoposide resistance, 29 of the 44 primary AML samples were cultured on stroma overnight, and activation of intracellular signaling pathways, including pY-STAT3, pY-STAT5, and pERK1/2, was measured by FACS. Responses were heterogeneous overall, but we found several patterns of stroma-induced signaling. For example, a sample that strongly activated a given pathway when co-cultured with HS5 also responded strongly to HS27A cells, and this was particularly true for pY-STAT3 (R=0.71, p<0.0001) and pY-STAT5 (R=0.76, p<0.0001). We also found a correlation between pY-STAT3 and pY-STAT5, such that samples that showed a strong activation of STAT3 with stromal co-culture had a similar activation of STAT5 (R=0.94, p<0.0001, on HS27A). As for signaling pathways in relationship to apoptosis, higher levels of pERK1/2 were associated with lower levels of apoptosis (R=-0.5182, p<0.01, on HS27A), suggesting that ERK1/2 activation may promote resistance to etoposide.

In summary, we found that DNA damage signaling is induced in AML cells by mitoxantrone, and it is augmented in cells co-cultured with stroma, likely contributing to mitoxantrone resistance. This mechanism does not occur with etoposide. Instead, we found that stromal environment-induced ERK1/2 signaling may enhance etoposide resistance in pediatric AML patient samples. Further studies to confirm the role of pERK1/2 in stroma-induced etoposide resistance are underway.